The present invention relates to an enzyme reaction method and a method for enzymatically producing an optically active cyanohydrin.
An enzyme reaction advantageously uses an organic solvent as a reaction solvent so as to increase the concentration of a substrate or product that is hard to dissolve in water. Such an enzyme reaction in an organic solvent has therefore been used as a reaction system for various enzymes. However, in contrast to an aqueous environment in which an enzyme is stable and active, an enzyme is often denatured and thus generally unstable in an organic solvent system. For this reason, in cases where enzyme reactions are applied to industrial syntheses of useful substances, the construction of reaction systems depends on the nature of enzymes to be used. A choice between high concentration reaction conditions and the stability of enzyme is therefore made according to individual circumstances. Particularly, in a method wherein hydroxynitrile lyase catalyzes the synthesis of an optically active cyanohydrin from hydrogen cyanide and a carbonyl compound as substrates, this enzyme is relatively stable in an organic solvent, but it provides a significant decrease in reaction rate when water is absent from the reaction system. Accordingly, such a conventionally known reaction using hydroxynitrile lyase in an organic solvent system involves a problem of extended reaction time. In addition, an aromatic carbonyl compound used as a substrate for this enzyme has low solubility in water, so that it is not practical to carry out such a reaction in an aqueous system due to low substrate and product concentrations.
With regard to the synthesis of an optically active cyanohydrin in the presence of hydroxynitrile lyase, the following reaction systems have been reported: an aqueous system, i.e., a system which uses water or an aqueous buffer containing an enzyme and a substrate dissolved therein (Japanese Patent Examined Publication No. 07-53116); a mixed solvent system comprising a mixture of a polar solvent and water (Appl. Microbiol. Biotechnol., Vol. 29, 419-425, 1988); an organic solvent system saturated with water or an aqueous buffer (Japanese Patent Laid-Open Publication No. 63-219388); and a two-phase system comprising a mixture of an organic solvent with water or an aqueous buffer at a volume ratio of 1/5 to 5/1 (Japanese Patent Laid-Open Publication No. 05-317065; Biocatal. Biotrans. Vol. 12, 255-266, 1995; Japanese Patent Laid-Open Publication No. 11-243983).
An aqueous reaction system involves a problem of insufficient efficiency attributed to low substrate and product concentrations because a carbonyl substrate, such as aldehyde or ketone, generally has low solubility in water. A mixed solvent system comprising a mixture of a polar solvent and water also involves a problem of insufficient efficiency although it provides some increases in substrate concentration, as compared with a simple aqueous system. This system involves an additional problem that a polar solvent is likely to affect the stability of the enzyme. The use of an organic solvent saturated with water or an aqueous buffer achieves higher substrate and product concentrations, but on the other hand, it provides a low reaction rate because the water content is too low. A two-phase system comprising an organic solvent and water is advantageous in improving a reaction rate and in increasing substrate and product concentrations. In practice, however, an enzyme comes into direct contact with an organic solvent in this system, so that the enzyme and contaminant proteins are likely to be denatured by the organic solvent. The denatured proteins may affect separation at the interface between organic and aqueous phases and, in some cases, may result in an emulsified reaction mixture. The two-phase system therefore involves difficulties of the separation of a reaction mixture into two phases.
An enzyme reaction using an aldehyde compound as a substrate is a widely carried out reaction. In particular, an enzyme reaction for synthesizing an optically active cyanohydrin from an aldehyde compound and hydrogen cyanide as substrates is useful because this reaction enables efficient synthesis of an optically active cyanohydrin which is difficult to chemically synthesize.
Thus, the above reaction is very advantageous, but it is known that, for example, the synthesis of (R)-mandelonitrile from benzaldehyde in the presence of (R)-hydroxynitrile lyase as a catalyst is inhibited when benzaldehyde as a starting material is contaminated by benzoic acid. However, there is no knowledge regarding acceptable concentration and effective removal of impurities such as benzoic acid, which act as inhibitors against enzyme reactions.
Impurities present in a starting aldehyde compound may be removed by distillation. However, distillation is not easily applied to industrial processes because it requires a distillation plant, it cannot completely separate some impurities such as benzoic acid due to their sublimation properties, and it may accelerate the production of carboxylic acids due to the application of heat. It has been therefore desirable to develop an enzyme reaction method that achieves simple and effective removal of impurities and thereby provides a product of interest in high yield.
There have been many reports about reactions for the synthesis of an optically active cyanohydrin from a carbonyl compound and prussic acid in the presence of an enzyme catalyst such as hydroxynitrile lyase. However, none of these reports has mention an industrially produced prussic acid containing a stabilizer which seriously affects the activity of hydroxynitrile lyase. There are two possible reasons for this. First, the previous reports took little notice of the above fact because prussic acid used therein was prepared for laboratory use in a very small amount, but not industrially produced, so that it contained no stabilizer. Second, the above fact did not present a problem in cases where prussic acid was added to a reaction system at low concentration the stabilizer hardly affected the reaction.
Further, when an optically active cyanohydrin is synthesized from a carbonyl compound and prussic acid (starting materials) in an organic solvent (reaction solvent) in the presence of hydroxynitrile lyase as a catalyst, the resulting reaction product is dissolved in the organic solvent used as a reaction solvent.
To collect the optically active cyanohydrin produced in this reaction, in general, there is a need to remove low-boiling solvents by distillation from the reaction product solution containing the optically active cyanohydrin. However, recycling of the distillate was previously unknown although collection of an optically active cyanohydrin through distillation of a reaction product solution is a known process.